Vitamin D is the general name for a collection of natural sterol-like substances including vitamin D2 and D3. As shown in FIG. 1, Vitamin D3 is synthesized in the skin from 7-dehydrocholesterol, a cholesterol breakdown product, via photochemical reactions using ultraviolet (UV) radiation from sunlight. The inert vitamin D3 is first converted to a largely inert intermediate by the liver to 25-HydroxyVitamin D3 (25-OH-D3) and then converted by the kidney to the bioactive hormone 1-25-DihydoxyVitamin D3 (1,25(OH)2D3) (FIG. 1). The bioactive vitamin D hormone, 1,25(OH)2D3, mediates its action by binding to vitamin D receptor (VDR) that is principally located in the nuclei of the target cell.
Vitamin D is a natural molecule that is biosynthesized by the interaction of sunlight with 7-dehydrocholesterol in the epidermis. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3 named calcitriol), the dihydroxylated metabolite of vitamin D3 is an essential nutrient for skeletal health. Calcitriol has profound effects on the growth and maturation of normal and malignant cells. Several epidemiological studies have demonstrated that people who live in higher latitudes are at higher risk of developing and dying of many cancers, including prostate cancer. It has also been demonstrated that there is an inverse relationship between latitude, sun-exposure and cutaneous synthesis of vitamin D (20). In 1989, Garland et al. carried out an eight-year prospective study among 26,520 healthy adults to demonstrate that if the initial level of serum of calcifediol [25-hydroxyvitamin D3 (25-OH-D3)], the mono-hydroxylated pre-hormonal form of calcitriol is at least 20 ng/ml, there is a 50% reduced risk of developing colon cancer. Since this observation other investigators have confirmed latitudinal impact and vitamin D intake on reducing risk of various cancers, including breast, prostate, renal and ovary. Vitamin D deficiency has also been correlated with autoimmune disease such as Multiple Sclerosis, hypertension, osteoporosis, bone diseases, rickets, psoriasis and infectious diseases.
Prostate cancer (PCA) is the most prevalent cancer among men; and the second leading cause of cancer death among men in the US. More than 500,000 PCA cases are diagnosed each year, 1 in 6 American males will develop PCA and 30,000 die each year in the US. Current clinical interventions for PCA include surgical removal of prostate and radiation therapy, with adverse side effects such as impotence, incontinence and alopecia. The mainstay of hormone-sensitive prostate cancer (HSPCA) chemotherapy is androgen-deprivation. After 9 to 30 months, HSPCA usually becomes insensitive to hormonal therapy and rapidly leads to HRPCA for which there are few interventions except for Sanofi-Aventis' Taxotere® (docetaxel) that has problems of toxicity and other adverse side-effects. New drugs have been recently approved for castration-resistant, docetaxel-refractory prostate cancers that extend life by 4.8 months.
Numerous studies have registered strong promise of calcitriol as a therapeutic agent for prostate and other cancers. However, its clinical use has been limited by risk of toxicity related to hypercalcemia, hypercalciuria, and significant loss of body weight. Attempts to address the toxicity-issue have taken two paths. In the first, combinations of calcitriol with standard chemotherapeutic agents are being investigated to harness synergy between these compounds. For example, clinical and animal studies have been carried out demonstrate that toxic effects of calcitriol can be mitigated by a combination with dexamethasone or paclitaxel.
Several attempts have been made to develop less/non-toxic analogs of calcitriol with potent antiproliferative activities as potential therapeutic agents. A Phase II clinical trial evaluated Seocalcitol (EB-1089), a side-chain analog of the active vitamin D hormone, in patients with inoperable pancreatic cancer. No objective responses (anti-tumor) activity was observed; the most frequent toxicity was dose-dependent hypercalcemia with most patients tolerating a dose of 10-15 μg/day in chronic administration.
The nuclear vitamin D receptor (VDR) plays a central role in the cell signaling process leading to anti-proliferation, and in some cases apoptosis of cancer cells. In this respect calcitriol is very similar to other steroidal and non-steroidal hormones such as estrogen, androgens, retinoids, glucocorticoids etc. Furthermore, VDR has high structural homology with nuclear receptors of other hormones. It is well established that cellular regulation by calcitriol and its analogs are initiated by highly specific binding to VDR, which is translated into pro-differentiation and concomitant antiproliferation of cells. Most human prostate cancer cells contain VDR; and numerous studies have shown that several prostate cancer cells respond to calcitriol. These findings strongly support the use of vitamin D-based agents for first line therapy and/or second line therapy when androgen deprivation fails.
However, cancer-therapy with calcitriol is limited by its rapid catabolic degradation by CYP-hydroxylases, which reduces its potency. As a result high doses of calcitriol are required clinically to harness its beneficial property; but such pharmacological doses cause toxicity. A way of circumventing this problem will be to covalently attach calcitriol into the ligand-binding pocket of VDR as shown in FIG. 2, so that (i) calcitriol is prevented from interacting with catabolic enzymes; and (ii) VDR-mediated transcriptional process could be set in motion since the ligand is inside the ligand-binding pocket of VDR leading to conformational changes required for the transcriptional process.
During the past decade hundreds of vitamin D analogs have been synthesized with the goal of obtaining a better antitumor/toxicity ratio and tumor-specific effect. Although a few of these analogs have successfully completed preclinical studies for several cancers; and at least one analog has recently failed Phase II clinical trials for pancreatic carcinomas, the majority of these compounds have been proved to be of limited therapeutic value due to toxicity. As a result new strategies for developing such analogs are required.
Aspects of the present invention employ materials known as supercritical, critical or near-critical fluids. A material becomes a critical fluid at conditions which equal its critical temperature and critical pressure. A material becomes a supercritical fluid at conditions which equal or exceed both its critical temperature and critical pressure. The parameters of critical temperature and critical pressure are intrinsic thermodynamic properties of all sufficiently stable pure compounds and mixtures. Carbon dioxide, for example, becomes a supercritical fluid at conditions which equal or exceed its critical temperature of 31.1° C. and its critical pressure of 72.8 atm (1,070 psig). In the supercritical fluid region, normally gaseous substances such as carbon dioxide become dense phase fluids which have been observed to exhibit greatly enhanced solvating power. At a pressure of 3,000 psig (204 atm) and a temperature of 40° C., carbon dioxide has a density of approximately 0.8 g/cc and behaves much like a nonpolar organic solvent, having a dipole moment of zero Debyes.
A supercritical fluid displays a wide spectrum of solvation power as its density is strongly dependent upon temperature and pressure. Temperature changes of tens of degrees or pressure changes by tens of atmospheres can change a compound solubility in a supercritical fluid by an order of magnitude or more. This feature allows for the fine-tuning of solvation power and the fractionation of mixed solutes. The selectivity of nonpolar supercritical fluid solvents can also be enhanced by addition of compounds known as modifiers (also referred to as entrainers or cosolvents). These modifiers are typically somewhat polar organic solvents such as acetone, ethanol, methanol, methylene chloride or ethyl acetate. Varying the proportion of modifier allows wide latitude in the variation of solvent power.
In addition to their unique solubilization characteristics, supercritical fluids possess other physicochemical properties which add to their attractiveness as solvents. They can exhibit liquid-like density yet still retain gas-like properties of high diffusivity and low viscosity. The latter increases mass transfer rates, significantly reducing processing times. Additionally, the ultra-low surface tension of supercritical fluids allows facile penetration into microporous materials, increasing extraction efficiency and overall yields.
A material at conditions that border its supercritical state will have properties that are similar to those of the substance in the supercritical state. These so-called “near-critical” fluids are also useful for the practice of this invention. For the purposes of this invention, a near-critical fluid is defined as a fluid which is (a) at a temperature between its critical temperature (Tc) and 75% of its critical temperature and at a pressure at least 75% of its critical pressure, or (b) at a pressure between its critical pressure (Pc) and 75% of its critical pressure and at a temperature at least 75% of its critical temperature. In this definition, pressure and temperature are defined on absolute scales, e.g., Kelvin and psia. To simplify the terminology, materials which are utilized under conditions which are supercritical, near-critical, or exactly at their critical point with or without polar co-solvents such as ethanol will jointly be referred to as “SuperFluids™” or referred to as “SFS.” SuperFluids™ were used for the nanoencapsulation of the Vitamin D analog in the protective lipid layer of phospholipid nanosomes.